The current research of our group is focused on the mechanism of action of ATP-dependent molecular chaperones and the role of chaperones in ATP-dependent proteolysis. Molecular chaperones are present in all organisms and are highly conserved. Chaperones participate in many and perhaps all cellular processes. Numerous chaperones are induced by environmental stresses such as heat shock, oxidative stress, and heavy metals, or pathologic conditions, such as inflammation, tissue damage, infection, and genetic diseases involving mutant proteins. They play a critical role during cell stress to prevent the appearance of folding intermediates that lead to irreversibly damaged proteins and assist in the recovery from stress either by refolding and reactivating damaged proteins or by disaggregating and unfolding damaged proteins and delivering them to compartmentalized proteases. The interrelationship between stress signaling, cell death, and oncogenesis has implicated the molecular chaperones as potential targets for cancer diagnosis and treatment. We previously found that Escherichia coli ClpA, an AAA+ ATPase and the regulatory component of ClpAP protease, has molecular chaperone activity. We are currently investigating how Clp proteins recognize specific substrates. The ClpA and ClpX ATPases associate with ClpP peptidase forming ClpAP and ClpXP proteases. Both ClpA and ClpX generally recognize short amino acid sequences located near the N- or C-terminus of a substrate. However, ClpAP and ClpXP are able to degrade proteins in which the end containing the recognition signal is fused to GFP such that the signal is in the interior of the primary sequence of the substrate. We tested if the internal ClpA recognition signal was the sole element required for targeting the substrate, RepA in this case, to ClpA. We discovered that, in the absence of a high affinity peptide recognition signal at the terminus, two elements are important for recognition of GFP-RepA fusion proteins by ClpA. One element is the natural ClpA recognition signal located at the junction of GFP and RepA in the fusion protein. The second is the C-terminal peptide of the fusion protein. Together these two elements facilitate binding and unfolding by ClpA and degradation by ClpAP. The internal site appears to function similarly to Clp adaptor proteins (proteins which facilitating degradation of specific substrates), but differs in that it is covalently attached to the polypeptide containing the terminal tag and both the "adaptor" and "substrate" modules are degraded. We have also been investigating how ClpX interacts with one of its adaptor proteins, RssB, a protein required to specifically promote degradation of the stationary phase RNA polymerase sigma factor, sigma S by ClpXP. By constructing and analyzing ClpX mutants and RssB mutants, we have discovered that amino acid residues in the C-terminal region of RssB are critical for ClpXP degradation of sigma S and for the interaction of RssB with ClpX. The C-terminal region of RssB is also necessary for the interaction of RssB with sigma S. RssB interacts with N-terminal domain of ClpX. Our results are revealing the pathway of protein interactions that take part in the regulation of sigma S stability. ClpB, another AAA+ member of the Clp chaperone family, is able to dissolve protein aggregates and reactivate proteins in conjunction with the DnaK chaperone and its two co-chaperones, DnaJ and GrpE. The mechanism for ClpB function in disaggregation is still undefined, but two models have been suggested. In one model DnaK, DnaJ and GrpE are proposed to loosen polypeptides on the surface of an aggregate and make them available for ClpB binding and unfolding. In a second model, the disaggregation ability of ClpB has been suggested to be via a crowbar mechanism, using the movements of a long coiled-coil domain of ClpB.